Mapping the dynamics of ligand reorganization via 13CH₃ and 13CH₂ relaxation dispersion at natural abundance

摘要

Flexible ligands pose challenges to standard structure-activity studies since they frequently reorganize their conformations upon protein binding and catalysis. Here, we demonstrate the utility of side chain 13C relaxation dispersion measurements to identify and quantify the conformational dynamics that drive this reorganization. The dispersion measurements probe methylene 13CH₂ and methyl 13CH₃ groups; the latter are highly prevalent side chain moieties in known drugs. Combining these side chain studies with existing backbone dispersion studies enables a comprehensive investigation of μs–ms conformational dynamics related to binding and catalysis. We perform these measurements at natural 13C abundance, in congruence with common pharmaceutical research settings. We illustrate these methods through a study of the interaction of a phosphopeptide ligand with the peptidyl-prolyl isomerase, Pin1. The results illuminate the side-chain moieties that undergo conformational readjustments upon complex formation. In particular, we find evidence that multiple exchange processes influence the side chain dispersion profiles. Collectively, our studies illustrate how side-chain relaxation dispersion can shed light on ligand conformational transitions required for activity, and thereby suggest strategies for its optimization.